Novel inhibitors of vascular endothelial growth factor activity, their uses and processes for their production

ABSTRACT

The present invention is directed to novel chimeric VEGF receptor proteins comprising amino acid sequences derived from the vascular endothelial growth factor (VEGF) receptors flt-1 and KDR, including the murine homologue to the human KDR receptor FLK-1, wherein said chimeric VEGF receptor proteins bind to VEGF and antagonize the endothelial cell proliferative and angiogenic activity thereof. The present invention is also directed to nucleic acids and expression vectors encoding these chimeric VEGF receptor proteins, host cells harboring such expression vectors, pharmaceutically acceptable compositions comprising such proteins, methods of preparing such proteins and to methods utilizing such proteins for the treatment of conditions associated with undesired vascularization.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of prior application Ser. No.11/537,623 filed Sep. 30, 2006, which is a continuation of ApplicationNo. 11,043,693 filed Jan. 26, 2005, which is a continuation ofapplication Ser. No. 10/105,901 filed Mar. 20, 2002, now U.S. Pat. No.6,897,294, which is a continuation of application Ser. No. 09/348,886filed Jul. 1, 1999, now U.S. Pat. No. 6,383,486, which is a divisionalof application Ser. No. 08/643,839 filed May 7, 1996, now U.S. Pat. No.6,100,071.

FIELD OF THE INVENTION

The present invention is directed to novel chimeric VEGF receptorproteins comprising amino acid sequences derived from the vascularendothelial growth factor (VEGF) receptors flt-1, KDR and the murinehomologue of the human KDR receptor, FLK-1, wherein said chimeric VEGFreceptor proteins bind to VEGF and antagonize the endothelial cellproliferative and angiogenic activity thereof. The present invention isalso directed to nucleic acids and expression vectors encoding thesechimeric VEGF receptor proteins, host cells harboring such expressionvectors, pharmaceutically acceptable compositions comprising suchproteins, methods of preparing such proteins and to methods utilizingsuch proteins for the treatment of conditions associated with undesiredvascularization.

BACKGROUND OF THE INVENTION

The two major cellular components of the mammalian vascular system arethe endothelial and smooth muscle cells. Endothelial cells form thelining of the inner surface of all blood vessels in the mammal andconstitute a non-thrombogenic interface between blood and tissue.Therefore, the proliferation of endothelial cells is an importantcomponent for the development of new capillaries and blood vesselswhich, in turn, is a necessary process for the growth and/orregeneration of mammalian tissues.

One protein that has been shown to play an extremely important role inpromoting endothelial cell proliferation and angiogenesis is vascularendothelial growth factor (VEGF). VEGF is a heparin-binding endothelialcell growth factor which was originally identified and purified frommedia conditioned by bovine pituitary follicular or folliculostellate(FS) cells. Ferrara and Henzel, Biochem. Biophys. Res. Comm. 161:851-858(1989). VEGF is a dimer with an apparent molecular mass of about 46 kDawith each subunit having an apparent molecular mass of about 23 kDa.Human VEGF is expressed in a variety of tissues as multiple homodimericforms (121, 165, 189 and 206 amino acids per monomer), wherein each formarises as a result of alternative splicing of a single RNA transcript.VEGF₁₂₁ is a soluble mitogen that does not bind heparin whereas thelonger forms of VEGF bind heparin with progressively higher affinity.

Biochemical analyses have shown that VEGF exhibits a strong mitogenicspecificity for vascular endothelial cells. For example, mediaconditioned by cells transfected by human VEGF cDNA promoted theproliferation of capillary endothelial cells, whereas medium conditionedby control cells did not. Leung, et al., Science 246:1306 (1989). Thus,VEGF is known to promote vascular endothelial cell proliferation andangiogenesis, a process which involves the formation of new bloodvessels from preexisting endothelium. As such, VEGF may be useful forthe therapeutic treatment of numerous conditions in which agrowth-promoting activity on the vascular endothelial cells isimportant, for example, in ulcers, vascular injuries and myocardialinfarction.

In contrast, however, while vascular endothelial proliferation isdesirable under certain circumstances, vascular endothelialproliferation and angiogenesis are also important components of avariety of diseases and disorders including tumor growth and metastasis,rheumatoid arthritis, psoriasis, atherosclerosis, diabetic retinopathy,retrolental fibroplasia, neovascular glaucoma, age-related maculardegeneration, hemangiomas, immune rejection of transplanted cornealtissue and other tissues, and chronic inflammation. Obviously, inindividuals suffering from any of these disorders, one would want toinhibit, or at least substantially reduce, the endothelial proliferatingactivity of the VEGF protein.

In the specific case of tumor cell growth, angiogenesis appears to becrucial for the transition from hyperplasia to neoplasia and forproviding nourishment to the growing solid tumor. Folkman, et al.,Nature 339:58 (1989). Angiogenesis also allows tumors to be in contactwith the vascular bed of the host, which may provide a route formetastasis of tumor cells. Evidence for the role of angiogenesis intumor metastasis is provided, for example, by studies showing acorrelation between the number and density of microvessels in histologicsections of invasive human breast carcinoma and actual presence ofdistant metastasis. Weidner et al., New Engl. J. Med. 324:1 (1991).Thus, one possible mechanism for the effective treatment of neoplastictumors is to inhibit or substantially reduce the endothelialproliferative and angiogenic activity of the VEGF protein.

The endothelial proliferative activity of VEGF is known to be mediatedby two high affinity tyrosine kinase receptors, flt-1 and KDR, whichexist only on the surface of vascular endothelial cells. DeVries,et-al., Science 225:989-991 (1992) and Terman, et al., Oncogene6:1677-1683 (1991). Both the flt-1 and KDR tyrosine kinase receptorshave seven immunoglobulin-like (Ig-like) domains which form theextracellular ligand-binding regions of the receptors, a transmembranedomain which serves to anchor the receptor on the surface of cells inwhich it is expressed and an intracellular catalytic tyrosine kinasedomain which is interrupted by a “kinase insert”. While the KDR receptorbinds only the VEGF protein with high affinity, the flt-1 receptor alsobinds placenta growth factor (PLGF), a molecule having significantstructural homology with VEGF. An additional member of the receptortyrosine kinases having seven Ig-like domains in the extracellularligand-binding region is FLT4, which is not a receptor for either VEGFor PLGF, but instead binds to a different ligand; VH1.4.5. The VH1.4.5ligand has been reported in the literature as VEGF-related protein (VRP)or VEGF-C.

Recent gene knockout studies have demonstrated that both the flt-1 andKDR receptors are essential for the normal development of the mammalianvascular system, although their respective roles in endothelial cellproliferation and differentiation appear to be distinct. Thus, theendothelial proliferative and angiogenic activity of the VEGF protein ismediated by binding to the extracellular ligand-binding region of theflt-1 and KDR receptors on the surface of vascular endothelial cells.

In view of the role of VEGF in vascular endothelial proliferation andangiogenesis, and the role that these processes play in many differentdiseases and disorders, it is desirable to have a means for reducing orinhibiting one or more of the biological activities of VEGF. As such,the present invention is predicated upon research intended to identifythe Ig-like domain or domains of the flt-1 and KDR receptorextracellular ligand-binding region which mediate binding to the VEGFprotein and inserting or fusing that domain or domains into amino acidsequences derived from another protein to produce a “chimeric VEGFreceptor protein”. The chimeric VEGF receptor proteins of the presentinvention will bind to and inactivate endogenous VEGF, thereby providinga means for reducing or inhibiting endogenous VEGF activity and, inturn, reducing or inhibiting endothelial cell proliferation andangiogenesis. Thus, it is an object of the present invention to providenovel chimeric VEGF receptor proteins comprising amino acid sequencesderived from the extracellular ligand-binding region of the flt-1 andKDR receptors, wherein said chimeric VEGF receptor proteins are capableof binding to and inhibiting the activity of VEGF.

Further objects of the present invention are to provide nucleic acidsencoding chimeric VEGF receptor proteins of the present invention,replicable expression vectors capable of expressing such chimericproteins, host cells transfected with those expression vectors,pharmaceutical compositions comprising the chimeric VEGF receptorproteins of the present invention, methods for preparing such chimericproteins and method of using those chimeric proteins for the therapeutictreatment of an individual in need thereof.

SUMMARY OF THE INVENTION

The objects of this invention, as generally defined supra, are achievedby the provision of chimeric VEGF receptor proteins which are capable ofbinding to VEGF and exerting an inhibitory effect thereon, wherein saidchimeric VEGF receptor protein comprises Ig-like domains 1, 2 and 3 ofthe flt-1 and/or the KDR receptor (or the murine homologue of the KDRreceptor, FLK-1) or functional equivalents thereof.

In a preferred embodiment, the chimeric VEGF receptor proteins of thepresent invention contain flt-1 or KDR receptor amino acid sequencescorresponding only to Ig-like domains 1, 2 and 3 of the extracellularligand-binding region thereof and each Ig-like domain is derived fromthe same VEGF receptor.

In other embodiments, however, the chimeric VEGF receptor proteins ofthe present invention comprise Ig-like domains 1, 2 and 3 of theextracellular ligand-binding region of the flt-1 or KDR receptor inaddition to one or more of the remaining four immunoglobulin-tikedomains thereof. Preferably, the Ig-like domains employed are derivedfrom the same receptor, however, a combination of Ig-like domainsderived from both the flt-1 and KDR receptors will find use.

In another embodiment of the present invention, the chimeric VEGFreceptor proteins of the present invention comprise the extracellularligand-binding region of the FLT4 receptor wherein at least Ig-likedomain 2 of the FLT4 receptor is replaced with the Ig-like domain 2 ofeither the flt-1 or KDR receptor. Preferably, only Ig-like domain 2 ofthe FLT4 receptor is replaced by the corresponding Ig-like domain fromeither the flt-1 or KDR receptor, however, other domains may also besimilarly replaced.

A further aspect of the present invention is directed to nucleic acidsequences encoding the chimeric VEGF receptor proteins described hereinand functional equivalents thereof. It is well known to the ordinarilyskilled artisan that such nucleic acids can vary due to the degeneracyof the genetic code and such nucleic acid variants are also encompassedby the present invention.

In still other embodiments, the present invention relates to replicableexpression vectors encoding the various chimeric VEGF receptor proteinsdescribed supra, host cells transfected with those expression vectorsand compositions comprising the chimeric VEGF receptor proteinsdescribed supra compounded with a pharmaceutically acceptable excipient.

In yet other embodiments, the present invention relates to methods forproducing the chimeric VEGF receptor proteins described supra byintroducing an expression vector encoding the desired chimeric proteininto an appropriate expression systems and effecting the expression ofsaid protein.

Yet another aspect of the invention provides for the use of the chimericVEGF receptor proteins of the present invention for the treatment ofconditions associated with inappropriate vascularization wherein aninhibition of vascularization and angiogenesis is desirable.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an alignment of the amino acid sequences for theextracellular ligand-binding regions of the flt-1, KDR and FLT4receptors. Amino acids are presented by their standard single letterdesignations. Dashes are inserted to create the best fit for alignmentof the seven Ig-like domains. The seven Ig-like domains of eachextracellular ligand-binding region of each receptor presented are shownas boxed areas.

FIG. 2 presents the oligonucleotides used in theoligonucleotide-directed mutagenesis-generated deletions of each of theseven Ig-like domains existing within the extracellular ligand-bindingregion of the flt-1 receptor. The restriction sites created in the DNAsequence are underlined and are listed above the underlined sequence.Amino acids are presented by their standard one-letter designations.Numbers shown in parentheses designate the amino acid number. In somecases, amino acids were changed when the restriction sites were designedinto the oligonucleotides. These amino acid changes are underlined andthe original amino acid at that position is presented below inparentheses.

FIG. 3 shows the ability of intact flt-1/IgG chimeric VEGF receptorprotein and various flt-1/IgG domain deletion chimeric proteins tospecifically bind to the VEGF ligand. Binding efficiency is presented asthe total cpm bound per 5 ng of immunoreactive (ir) F_(e). “wt” refersto the intact flt-1/IgG chimeric VEGF receptor protein. “d1” through“d7” refer to the flt-1/IgG domain deletion chimeras where the numbercorresponds to the Ig-like domain that is deleted.

FIG. 4 shows the ability of intact flt-1/IgG chimeric VEGF receptorprotein and various flt-1/IgG domain deletion chimeric proteins tospecifically bind to the VEGF ligand. Binding efficiency is presented asthe total cpm bound per 4.5 ng of immunoreactive (ir) F_(c). “flt-wt”refers to the intact flt-1/IgG chimeric protein. “flt(1,2)” is thechimeric protein having only flt-1 Ig-like domains 1 and 2 fused to theF_(c) of IgG. “flt(2)” is the chimeric protein having only flt-1 Ig-likedomain 2 fused to the F_(c) of IgG. “flt(2,3)” is the chimeric proteinhaving only flt-1 Ig-like domains 2 and 3 fused to the F_(c) of IgG.“flt(1,2,3)” is the chimeric protein having only flt-1 Ig-like domains1, 2 and 3 fused to the F_(c) of IgG. Finally, “KDR(2)” is a chimericVEGF receptor protein wherein only the Ig-like domain 2 of theextracellular ligand-binding region of the KDR receptor is fused to theF_(c) of IgG.

FIG. 5 shows the percent binding of the VEGF ligand to the intactflt-1/IgG chimeric VEGF receptor protein and to the flt(1,2,3) deletionchimera in the presence of increasing amounts of unlabeled VEGFcompetitor. “” designates binding by the flt(1,2,3) deletion chimera.“♦” designates binding by the intact flt-1/IgG chimeric protein.

FIG. 6 shows the ability of intact flt-1/IgG chimeric VEGF receptorprotein and various Ig-like domain 2 “swap” mutants to specifically bindto the VEGF ligand. Binding efficiency is presented as the total cpmbound per 1 ng of immunoreactive (ir) F_(c). “flt-1” refers to thenative flt-1/IgG chimeric protein. “flt.d2” refers to the flt-1/IgGchimeric protein having a deletion of Ig-like domain 2. “flt.K2” refersto the “swap” chimera protein where the Ig-like domain 2 of theflt-1/IgG protein is replaced with the Ig-like domain 2 of the KDRreceptor. Finally, “fltF4.2” refers to the “swap” chimera protein wherethe Ig-like domain 2 of the flt-1 protein is replaced with the Ig-likedomain 2 of the FLT4 receptor.

FIG. 7 shows the percent inhibition of VEGF binding by either unlabeledVEGF competitor or unlabeled PLGF competitor with various flt-1/IgG“swap” chimeric proteins. “flt” and “KDR” designate the native flt-1 andnative KDR receptor, respectively. “flt.K1” refers to the “swap” chimerawherein the Ig-like domain 1 of the flt-1 receptor is replaced by theIg-like domain 1 of the KDR receptor. “flt.K2” refers to the “swap”chimera wherein the Ig-like domain 2 of the flt-1 receptor is replacedby the Ig-like domain 2 of the KDR receptor. “flt.K3” refers to the“swap” chimera wherein the Ig-like domain 3 of the flt-1 receptor isreplaced by the Ig-like domain 3 of the KDR receptor. “flt.K5” refers tothe “swap” chimera wherein the Ig-like domain 5 of the flt-1 receptor isreplaced by the Ig-like domain 5 of the KDR receptor. Finally, “flt.K7”refers to the “swap” chimera wherein the Ig-like domain 7 of the flt-1receptor is replaced by the Ig-like domain 7 of the KDR receptor.

FIG. 8 shows the entire amino acid sequence of the intact FLT4 receptor.Amino acid residues are presented in their standard one-letterdesignations.

FIG. 9 shows the entire amino acid sequence of the receptor encoded bythe flt-1(1,2,3)/FLT4 expression construct. Underlined amino acidresidues are those derived from Ig-like domains 1-3 of the flt-1receptor and which replace the Ig-like domains 1-3 of the FLT4 receptor.The bolded amino acid residue differs from the wild type FLT4 amino acidresidue normally at that position. Amino acid residues are presented intheir standard one-letter designations.

FIG. 10 shows the entire amino acid sequence of the receptor encoded bythe flt-1(2)/FLT4 expression construct. Underlined amino acid residuesare those derived from Ig-like domain 2 of the flt-1 receptor and whichreplace the Ig-like domain 2 of the FLT4 receptor. The bolded amino acidresidues differ from the wild type FLT4 amino acid residues normally atthat position. Amino acid residues are presented in their standardone-letter designations.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

As used herein, the term “chimeric VEGF receptor protein” means areceptor molecule having amino acid sequences derived from at least twodifferent proteins, at least one of which is the flt-1 or KDR receptor,said receptor molecule being capable of binding to and inhibiting theactivity of VEGF. Preferably, the chimeric VEGF receptor proteins of thepresent invention consist of amino acid sequences derived from only twodifferent VEGF receptor molecules, however, amino acid sequencescomprising Ig-like domains from the extracellular ligand-binding regionof the flt-1 and/or KDR receptor can be linked to amino acid sequencesfrom other unrelated proteins, for example, immunoglobulin sequences.Other amino acid sequences to which Ig-like domains are combined will bereadily apparent to those of ordinary skill in the art.

The term “KDR receptor” as used herein is meant to encompass not onlythe KDR receptor but also the murine homologue of the human KDRreceptor, designated FLK-1.

“Immunoglobulin-like domain” or “Ig-like domain” refers to each of theseven independent and distinct domains that are found in theextracellular ligand-binding region of the flt-1, KDR and FLT4receptors. Ig-like domains are generally referred to by number, thenumber designating the specific domain as it is shown in FIG. 1. As usedherein, the term “Ig-like domain” is intended to encompass not only thecomplete wild-type domain, but also insertional, deletional andsubstitutional variants thereof which substantially retain thefunctional characteristics of the intact domain. It will be readilyapparent to those of ordinary skill in the art that numerous variants ofthe Ig-like domains of the flt-1 and KDR receptors can be obtained whichwill retain substantially the same functional characteristics as thewild type domain.

“Soluble” as used herein with reference to the chimeric VEGF receptorproteins of the present invention is intended to mean chimeric VEGFreceptor proteins which are not fixed to the surface of cells via atransmembrane domain. As such, soluble forms of the chimeric VEGFbinding proteins of the present invention, while capable of binding toand inactivating VEGF, do not comprise a transmembrane domain and thusgenerally do not become associated with the cell membrane of cells inwhich the molecule is expressed. A soluble form of the receptor exertsan inhibitory effect on the biological activity of the VEGF protein bybinding to VEGF, thereby preventing it from binding to its naturalreceptors present on the surface of target cells.

“Membrane-bound” as used herein with reference to the chimeric VEGFreceptor proteins of the present invention is intended to mean chimericVEGF receptor proteins which are fixed, via a transmembrane domain, tothe surface of cells in which they are expressed.

“Functional equivalents” when used in reference to the Ig-like domainsof the extracellular ligand-binding regions of the flt-1, KDR or FLT4receptors means the Ig-like domain or domains possess at least oneparticular alteration, such as a deletion, addition and/or substitutiontherein yet retains substantially the same functional characteristics asdoes the wild type Ig-like domain or domains with reference morespecifically to Ig-like domains 1, 2 and 3 of the flt-1 and/or KDRreceptor, “functional equivalents” intends scope of so much of suchdomains as to result in at least substantial binding to VEGF, i.e., apartial sequence of each of said domains that will produce a bindingeffect.

“Inhibitory effect” when used in reference to the activity of a chimericVEGF receptor protein of the present invention means that the chimericVEGF receptor protein binds to and substantially inhibits the activityof VEGF. Generally, the result of this inhibitory effect is a decreasein the vascularization and/or angiogenesis which occurs as a result ofthe VEGF protein.

“Undesired vascularization” refers to the endothelial proliferationand/or angiogenesis which is associated with an undesirable disease ordisorder and which, if reduced or eliminated, would result in areduction or elimination of the undesirable characteristics of thedisease or disorder. For example, the vascularization and/orangiogenesis associated with tumor formation and metastasis and variousretinopathies is undesirable.

“Transfection” refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Successful transfection is generallyrecognized when any indication of the operation of this vector occurswithin the host cell.

“Transformation” means introducing DNA into an organism so that the DNAis replicable, either as an extrachromosomal element or by chromosomalintegration. Depending on the host cell used, transformation is doneusing standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride, as described by Cohen, S. N.,Proc. Natl. Acad. Sci. (USA), 69, 2110 (1972) and Mandel et al. J. Mol.Biol. 53, 154 (1970), is generally used for prokaryotes or other cellsthat contain substantial cell-wall barriers. For mammalian cells withoutsuch cell walls, the calcium phosphate precipitation method of Graham,F. and van der Eb, A., Virology, 52, 456-457 (1978) is preferred.General aspects of mammalian cell host system transformations have beendescribed by Axel in U.S. Pat. No. 4,399,216 issued Aug. 16, 1983.Transformations into yeast are typically carried out according to themethod of Van Solingen, P., at al. J. Bact., 130, 946 (1977) and Hsiao,C. L., at al. Proc. Natl. Acad. Sci (USA) 76, 3829 (1979). However,other methods for introducing DNA into cells such as by nuclearinjection, electroporation or by protoplast fusion may also be used.

“Site-directed mutagenesis” is a technique standard in the art, and isconducted using a synthetic oligonucleotide primer complementary to asingle-stranded phage DNA to be mutagenized except for limitedmismatching, representing the desired mutation. Briefly, the syntheticoligonucleotide is used as a primer to direct synthesis of a strandcomplementary to the phage, and the resulting double-stranded DNA istransformed into a phage-supporting host bacterium. Cultures of thetransformed bacteria are plated in top agar, permitting plaque formationfrom single cells that harbor the phage. Theoretically, 50% of the newplaques will contain the phage having, as a single strand, the mutatedform; 50% will have the original sequence. The plaques are hybridizedwith kinased synthetic primer at a temperature that permitshybridization of an exact match, but at which the mismatches with theoriginal strand are sufficient to prevent hybridization. Plaques thathybridize with the probe are then selected and cultured, and the DNA isrecovered.

“Operably linked” refers to juxtaposition such that the normal functionof the components can be performed. Thus, a coding sequence “operablylinked” to control sequences refers to a configuration wherein thecoding sequence can be expressed under the control of these sequencesand wherein the DNA sequences being linked are contiguous and, in thecase of a secretory leader, contiguous and in reading phase. Forexample, DNA for a presequence or secretory leader is operably linked toDNA for a polypeptide if it is expressed as a preprotein thatparticipates in the secretion of the polypeptide; a promoter or enhanceris operably linked to a coding sequence if it affects the transcriptionof the sequence; or a ribosome binding site is operably linked to acoding sequence if it is positioned so as to facilitate translation.Linking is accomplished by ligation at convenient restriction sites. Ifsuch sites do not exist, then synthetic oligonucleotide adaptors orlinkers are used in accord with conventional practice.

“Control sequences” refers to DNA sequences necessary for the expressionof an operably linked coding sequence in a particular host organism. Thecontrol sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, a ribosome bindingsite, and possibly, other as yet poorly understood sequences. Eukaryoticcells are known to utilize promoters, polyadenylation signals, andenhancers.

“Expression system” refers to DNA sequences containing a desired codingsequence and control sequences in operable linkage, so that hoststransformed with these sequences are capable of producing the encodedproteins. To effect transformation, the expression system may beincluded on a vector; however, the relevant DNA may then also beintegrated into the host chromosome.

As used herein, “cell,” “cell line,” and “cell culture” are usedinterchangeably and all such designations include progeny. Thus,“transformants” or “transformed cells” includes the primary subject celland cultures derived therefrom without regard for the number oftransfers. It is also understood that all progeny may not be preciselyidentical in DNA content, due to deliberate or inadvertent mutations.Mutant progeny that have the same functionality as screened for in theoriginally transformed cell are included. Where distinct designationsare intended, it will be clear from the context.

“Plasmids” are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein arecommercially available, are publicly available on an unrestricted basis,or can be constructed from such available plasmids in accord withpublished procedures. In addition, other equivalent plasmids are knownin the art and will be apparent to the ordinary artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with anenzyme that acts only at certain locations in the DNA. Such enzymes arecalled restriction enzymes, and the sites for which each is specific iscalled a restriction site. The various restriction enzymes used hereinare commercially available and their reaction conditions, cofactors, andother requirements as established by the enzyme suppliers are used.Restriction enzymes commonly are designated by abbreviations composed ofa capital letter followed by other letters representing themicroorganism from which each restriction enzyme originally was obtainedand then a number designating the particular enzyme. In general, about 1mg of plasmid or DNA fragment is used with about 1-2 units of enzyme inabout 20 ml of buffer solution. Appropriate buffers and substrateamounts for particular restriction enzymes are specified by themanufacturer. Incubation of about 1 hour at 37° C. is ordinarily used,but may vary in accordance with the supplier's instructions. Afterincubation, protein is removed by extraction with phenol and chloroform,and the digested nucleic acid is recovered from the aqueous fraction byprecipitation with ethanol. Digestion with a restriction enzymeinfrequently is followed with bacterial alkaline phosphatase hydrolysisof the terminal 5′ phosphates to prevent the two restriction cleavedends of a DNA fragment from “circularizing” or forming a closed loopthat would impede insertion of another DNA fragment at the restrictionsite. Unless otherwise stated, digestion of plasmids is not followed by5′ terminal dephosphorylation. Procedures and reagents fordephosphorylation are conventional (T. Maniatis et al. 1982, MolecularCloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory,1982) pp. 133-134).

“Recovery” or “isolation” of a given fragment of DNA from a restrictiondigest means separation of the digest on polyacrylamide or agarose gelby electrophoresis, identification of the fragment of interest bycomparison of its mobility versus that of marker DNA fragments of knownmolecular weight, removal of the gel section containing the desiredfragment, and separation of the gel from DNA. This procedure is knowngenerally. For example, see R. Lawn et al., Nucleic Acids Res. 9,6103-6114 (1981) and D. Goeddel et al., Nucleic Acids Res. 8, 4057(1980).

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (T. Maniatis et al. 1982,supra, p. 146). Unless otherwise provided, ligation may be accomplishedusing known buffers and conditions with 10 units of T4 DNA ligase(“ligase”) per 0.5 mg of approximately equimolar amounts of the DNAfragments to be ligated.

“Preparation” of DNA from transformants means isolating plasmid DNA frommicrobial culture. Unless otherwise provided, the alkaline/SDS method ofManiatis et al. 1982, supra, p. 90, may be used.

“Oligonucleotides” are short-length, single- or double-strandedpolydeoxynucleotides that are chemically synthesized by known methods(such as phosphotriester, phosphite, or phosphoramidite chemistry, usingsolid phase techniques such as described in EP Pat. Pub. No. 266,032published May 4, 1988, or via deoxynucleoside H-phosphonateintermediates as described by Froehler et al., Nucl. Acids Res. 14,5399-5407 [1986]). They are then purified on polyacrylamide gels.

B. General Methodology

1. Amino Acid Sequence Variants

It will be appreciated that various amino acid substitutions can be madein the Ig-like domain or domains of the chimeric VEGF receptor proteinsof the present invention without departing from the spirit of thepresent invention with respect to the chimeric proteins' ability to bindto and inhibit the activity of VEGF. Thus, point mutational and otherbroader variations may be made in the Ig-like domain or domains of thechimeric VEGF receptor proteins of the present invention so as to impartinteresting properties that do not substantially effect the chimericprotein's ability to bind to and inhibit the activity of VEGF. Thesevariants may be made by means generally known well in the art.

a. Covalent Modifications

Covalent modifications may be made to various amino acid residues of theIg-like domain or domains present in the chimeric VEGF receptor protein,thereby imparting new properties to that Ig-like domain or domainswithout eliminating the capability to bind to and inactivate VEGF.

For example, cysteinyl residues most commonly are reacted witha-haloacetates (and corresponding amines), such as chloroacetic acid orchloroacetamide, to give carboxymethyl or carboxyamidomethylderivatives. Cysteinyl residues also are derivatized by reaction withbromotrifluoroacetone, a-bromo-b-(5-imidozoyl)propionic acid,chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide,methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing a-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues per se has been studiedextensively, with particular interest in introducing spectral labelsinto tyrosyl residues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizol and tetranitromethaneare used to form O-acetyl tyrosyl species and 3-nitro derivatives,respectively. Tyrosyl residues are iodinated using ¹²⁵I or ¹³¹I toprepare labeled proteins for use in radioimmunoassay, the chloramine Tmethod described above being suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′—N—C—N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinking thechimeric VEGF receptor protein to a water-insoluble support matrix orsurface for use in the method for purifying the VEGF protein fromcomplex mixtures. Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis-(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the a-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 [1983]),acetylation of the N-terminal amine, and, in some instances, amidationof the C-terminal carboxyl group.

b. DNA Mutations

Amino acid sequence variants of the Ig-like domain or domains present inthe chimeric VEGF receptor proteins of the present invention can also beprepared by creating mutations in the DNA encoding the chimeric protein.Such variants include, for example, deletions from, or insertions orsubstitutions of, amino acid residues within the amino acid sequence ofthe Ig-like domain or domains. Any combination of deletion, insertion,and substitution may also be made to arrive at the final construct,provided that the final construct possesses the desired activity.Obviously, the mutations that will be made in the DNA encoding thevariant must not place the sequence out of reading frame and preferablywill not create complementary regions that could produce secondary mRNAstructure (see EP 75,444A).

At the genetic level, variants of the Ig-like domain or domains presentin the chimeric VEGF receptor proteins of the present inventionordinarily are prepared by site-directed mutagenesis of nucleotides inthe DNA encoding the Ig-like domain or domains, thereby producing DNAencoding the variant, and thereafter expressing the DNA in recombinantcell culture. The variants typically exhibit the same qualitativeability to bind to the VEGF ligand as does the unaltered chimericprotein.

While the site for introducing an amino acid sequence variation in theIg-like domain or domains of the chimeric VEGF receptor protein ispredetermined, the mutation per se need not be predetermined. Forexample, to optimize the performance of a mutation at a given site,random mutagenesis may be conducted at the target codon or region andthe expressed chimeric protein variants screened for the optimalcombination of desired attributes such as ability to specifically bindto the VEGF ligand, in vivo half-life, and the like. Techniques formaking substitution mutations at predetermined sites in DNA having aknown sequence are well known, for example, site-specific mutagenesis.

Preparation of variants in the Ig-like domain or domains of a chimericVEGF receptor protein in accordance herewith is preferably achieved bysite-specific mutagenesis of DNA that encodes an earlier preparedchimeric protein. Site-specific mutagenesis allows the production ofIg-like domain variants through the use of specific oligonucleotidesequences that encode the DNA sequence of the desired mutation, as wellas a sufficient number of adjacent nucleotides, to provide a primersequence of sufficient size and sequence complexity to form a stableduplex on both sides of the deletion junction being traversed.Typically, a primer of about 20 to 25 nucleotides in length ispreferred, with about 5 to 10 residues on both sides of the junction ofthe sequence being altered. In general, the technique of site-specificmutagenesis is well known in the art, as exemplified by publicationssuch as Adelman et al., DNA 2, 183 (1983), the disclosure of which isincorporated herein by reference.

As will be appreciated, the site-specific mutagenesis techniquetypically employs a phage vector that exists in both a single-strandedand double-stranded form. Typical vectors useful in site-directedmutagenesis include vectors such as the M13 phage, for example, asdisclosed by Messing et al., Third Cleveland Symposium on Macromoleculesand Recombinant DNA, Editor A. Walton, Elsevier, Amsterdam (1981), thedisclosure of which is incorporated herein by reference. These phage arereadily commercially available and their use is generally well known tothose skilled in the art. Alternatively, plasmid vectors that contain asingle-stranded phage origin of replication (Veira at al., Meth.Enzymol., 153, 3 [1987]) may be employed to obtain single-stranded DNA.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector that includeswithin its sequence a DNA sequence that encodes the relevant chimericVEGF receptor protein. An oligonucleotide primer bearing the desiredmutated sequence is prepared, generally synthetically, for example, bythe method of Crea et al., Proc. Natl. Acad. Sci. (USA), 75, 5765(1978). This primer is then annealed with the single-stranded chimericprotein-sequence-containing vector, and subjected to DNA-polymerizingenzymes such as E. coli polymerase I Klenow fragment, to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells such as JM101 cells and clonesare selected that include recombinant vectors bearing the mutatedsequence arrangement.

After such a clone is selected, the mutated DNA encoding the variantchimeric VEGF receptor protein may be removed and placed in anappropriate vector for protein production, generally an expressionvector of the type that may be employed for transformation of anappropriate host.

c. Types of Mutations

Amino acid sequence deletions generally range from about 1 to 15residues, more preferably 1 to 7 residues, and typically are contiguous.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions of from one residue to polypeptides of essentially unrestrictedlength, as well as intrasequence insertions of single or multiple aminoacid residues. Intrasequence insertions (i.e., insertions within theIg-like domain sequences) may range generally from about 1 to 10residues, more preferably 1 to 5. An example of a terminal insertionincludes a fusion of a signal sequence, whether heterologous orhomologous to the host cell, to the N-terminus of the chimeric VEGFreceptor protein to facilitate the secretion of the chimeric proteinfrom recombinant hosts.

The third group of mutations which can be introduced into the Ig-likedomain or domains present in the chimeric VEGF receptor protein arethose in which at least one amino acid residue in the Ig-like domain ordomains, and preferably only one, has been removed and a differentresidue inserted in its place. Such substitutions preferably are made inaccordance with the following Table 1 when it is desired to modulatefinely the characteristics of the Ig-like domain or domains.

TABLE 1 Original Residue Exemplary Substitutions Ala (A) gly; ser Arg(R) lys Asn (N) gln; his Asp (D) glu Cys (C) ser Gln (Q) asn Glu (E) aspGly (G) ala; pro His (H) asn; gln Ile (I) leu; val Leu (L) ile; val Lys(K) arg; gln; glu Met (M) leu; tyr; ile Phe (F) met; leu; tyr Ser (S)thr Thr (T) ser Trp (W) tyr Tyr (Y) trp; phe Val (V) ile; leuSubstantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those in TableI, i.e., selecting residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example, as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the bulk of the side chain. The substitutions thatin general are expected to produce the greatest changes in theproperties of the Ig-like domains will be those in which (a) glycineand/or proline (P) is substituted by another amino acid or is deleted orinserted; (b) a hydrophilic residue, e.g., seryl or threonyl, issubstituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl,phenylalanyl, valyl, or alanyl; (c) a cysteine residue is substitutedfor (or by) any other residue; (d) a residue having an electropositiveside chain, e.g., lysyl, arginyl, or histidyl, is substituted for (orby) a residue having an electronegative charge, e.g., glutamyl oraspartyl; (e) a residue having an electronegative side chain issubstituted for (or by) a residue having an electropositive charge; or(f) a residue having a bulky side chain, e.g., phenylalanine, issubstituted for (or by) one not having such a side chain, e.g., glycine.

Most deletions and insertions, and substitutions in particular, are notexpected to produce radical changes in the characteristics of theIg-like domain or domains of the chimeric VEGF receptor protein.However, when it is difficult to predict the exact effect of thesubstitution, deletion, or insertion in advance of doing so, one skilledin the art will appreciate that the effect will be evaluated by routinescreening assays. For example, an Ig-like domain variant typically ismade by site-specific mutagenesis of the nucleic acid encoding theintact chimeric VEGF receptor protein, expression of the variant nucleicacid in recombinant cell culture, purification of the variant chimericVEGF receptor protein from the cell culture and detecting the ability ofthe variant chimeric VEGF receptor protein to specifically bind to aVEGF ligand. Binding assays which can be routinely employed to determineif a particular alteration or alterations in an Ig-like domain ordomains affects the capability of the chimeric VEGF receptor protein tobind to and inhibit the activity of VEGF are described both in theExamples below and in the article by Park et al., J. Biol. Chem.269:25646-25654 (1994) which is expressly incorporated by referenceherein.

Thus, the activity of a variant chimeric VEGF receptor protein may bescreened in a suitable screening assay for the desired characteristic.For example, a change in the ability to specifically bind to a VEGFligand can be measured by a competitive-type VEGF binding assay.Modifications of such protein properties as redox or thermal stability,hydrophobicity, susceptibility to proteolytic degradation, or thetendency to aggregate with carriers or into multimers are assayed bymethods well known to the ordinarily skilled artisan.

2. Recombinant Expression

The chimeric VEGF receptor proteins of the present invention areprepared by any technique, including by well known recombinant methods.Likewise, an isolated DNA is understood herein to mean chemicallysynthesized DNA, cDNA, chromosomal, or extrachromosomal DNA with orwithout the 3′- and/or 5′-flanking regions. Preferably, the desiredchimeric VEGF receptor protein herein is made by synthesis inrecombinant cell culture.

For such synthesis, it is first necessary to secure nucleic acid thatencodes a chimeric VEGF receptor protein of the present invention. DNAencoding a flt-1 or KDR receptor may be obtained from vascularendothelial cells by (a) preparing a cDNA library from these cells, (b)conducting hybridization analysis with labeled DNA encoding the flt-1 orKDR receptor or fragments thereof (up to or more than 100 base pairs inlength) to detect clones in the library containing homologous sequences,and (c) analyzing the clones by restriction enzyme analysis and nucleicacid sequencing to identify full-length clones. If full-length clonesare not present in a cDNA library, then appropriate fragments may berecovered from the various clones using the nucleic acid and amino acidsequence information known for the flt-1 and KDR receptors and ligatedat restriction sites common to the clones to assemble a full-lengthclone encoding the flt-1 or KDR domain. Alternatively, genomic librariesmay provide the desired DNA.

Once this DNA has been identified and isolated from the library, thisDNA may be ligated into an appropriate expression vector operablyconnected to appropriate control sequences. Moreover, once cloned intoan appropriate vector, the DNA can be altered in numerous ways asdescribed above to produce functionally equivalent variants thereof.Additionally, DNA encoding various domains, such as the intracellular,transmembrane and/or various Ig-like domains can be deleted and/orreplaced by DNA encoding corresponding domains from other receptors. DNAencoding unrelated amino acid sequences, such as the F_(c) portion of animmunoglobulin molecule, may also be fused to the DNA encoding some orall of the VEGF receptor, thereby producing a chimeric VEGF receptormolecule.

In one example of a recombinant expression system, an Ig-like domaincontaining chimeric VEGF receptor protein is expressed in mammaliancells by transformation with an expression vector comprising DNAencoding the chimeric VEGF receptor protein. It is preferable totransform host cells capable of accomplishing such processing so as toobtain the chimeric protein in the culture medium or periplasm of thehost cell, i.e., obtain a secreted molecule.

a. Useful Host Cells and Vectors

The vectors and methods disclosed herein are suitable for use in hostcells over a wide range of prokaryotic and eukaryotic organisms.

In general, of course, prokaryotes are preferred for the initial cloningof DNA sequences and construction of the vectors useful in theinvention.

For example, E. coli K12 strain MM 294 (ATCC No. 31,446) is particularlyuseful. Other microbial strains that may be used include E. coli strainssuch as E. coli B and E. coli X1776 (ATCC No. 31,537). These examplesare, of course, intended to be illustrative rather than limiting.

Prokaryotes may also be used for expression. The aforementioned strains,as well as E. coli strains W3110 (F-, lambda-, prototrophic, ATCC No.27,325), K5772 (ATCC No. 53,635), and SR101, bacilli such as Bacillussubtilis, and other enterobacteriaceae such as Salmonella typhimurium orSerratia marcesans, and various pseudomonas species, may be used.

In general, plasmid vectors containing replicon and control sequencesthat are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences that are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies (see, e.g., Bolivar et al., Gene 2, 95 [1977]). pBR322 containsgenes for ampicillin and tetracycline resistance and thus provides easymeans for identifying transformed cells. The pBR322 plasmid, or othermicrobial plasmid or phage, must also contain, or be modified tocontain, promoters that can be used by the microbial organism forexpression of its own proteins.

Those promoters most commonly used in recombinant DNA constructioninclude the β-lactamase (penicillinase) and lactose promoter systems(Chang et al., Nature, 375, 615 [1978]; Itakura at al., Science, 198,1056 [1977]; Goeddel et al., Nature, 281, 544 [1979]) and a tryptophan(trp) promoter system (Goeddel et al., Nucleic Acids Res., 8, 4057[1980]; EPO Appl. Publ. No. 0036,776). While these are the most commonlyused, other microbial promoters have been discovered and utilized, anddetails concerning their nucleotide sequences have been published,enabling a skilled worker to ligate them functionally with plasmidvectors (see, e.g., Siebenlist et al., Cell, 20, 269 [1980]).

In addition to prokaryotes, eukaryotic microbes, such as yeast cultures,may also be used. Saccharomyces cerevisiae, or common baker's yeast, isthe most commonly used among eukaryotic microorganisms, although anumber of other strains are commonly available. For expression inSaccharomyces, the plasmid YRp7, for example (Stinchcomb et al., Nature282, 39 [1979]; Kingsman at al., Gene 7, 141 [1979]; Tschemper at al.,Gene. 10, 157 [1980]), is commonly used. This plasmid already containsthe trp1 gene that provides a selection marker for a mutant strain ofyeast lacking the ability to grow in tryptophan, for example, ATCC No.44,076 or PEP4-1 (Jones, Genetics, 85, 12 [1977]). The presence of thetrp1 lesion as a characteristic of the yeast host cell genome thenprovides an effective environment for detecting transformation by growthin the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255, 2073[1980]) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7,149 [1968]; Holland at al., Biochemistry 17, 4900 [1978]), such asenolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In constructing suitableexpression plasmids, the termination sequences associated with thesegenes are also ligated into the expression vector 3′ of the sequencedesired to be expressed to provide polyadenylation of the mRNA andtermination. Other promoters, which have the additional advantage oftranscription controlled by growth conditions, are the promoter regionfor alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism, and theaforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization. Any plasmid vectorcontaining yeast-compatible promoter, origin of replication andtermination sequences is suitable.

In addition to microorganisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. However, interest has been greatest in vertebrate cells, andpropagation of vertebrate cells in culture (tissue culture) has become aroutine procedure in recent years [Tissue Culture, Academic Press, Kruseand Patterson, editors (1973)]. Examples of such useful host cell linesare VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, andW138, BHK, COS-7, 293, and MDCK cell lines. Expression vectors for suchcells ordinarily include (if necessary) an origin of replication, apromoter located in front of the gene to be expressed, along with anynecessary ribosome binding sites, RNA splice sites, polyadenylationsites, and transcriptional terminator sequences.

For use in mammalian cells, the control functions on the expressionvectors are often provided by viral material. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, and most frequentlySimian Virus 40 (SV40). The early and late promoters of SV40 virus areparticularly useful because both are obtained easily from the virus as afragment that also contains the SV40 viral origin of replication [Fierset al., Nature, 273, 113 (1978)]. Smaller or larger SV40 fragments mayalso be used, provided there is included the approximately 250-bpsequence extending from the Hind111 site toward the BgII site located inthe viral origin of replication. Further, it is also possible, and oftendesirable, to utilize promoter or control sequences normally associatedwith the desired gene sequence, provided such control sequences arecompatible with the host cell systems.

An origin of replication may be provided either by construction of thevector to include an exogenous origin, such as may be derived from SV40or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or may beprovided by the host cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosome, the latter is oftensufficient.

Satisfactory amounts of protein are produced by cell cultures; however,refinements, using a secondary coding sequence, serve to enhanceproduction levels even further. One secondary coding sequence comprisesdihydrofolate reductase (DHFR) that is affected by an externallycontrolled parameter, such as methotrexate (MTX), thus permittingcontrol of expression by control of the methotrexate concentration.

In selecting a preferred host cell for transfection by the vectors ofthe invention that comprise DNA sequences encoding both chimeric proteinand DHFR protein, it is appropriate to select the host according to thetype of DHFR protein employed. If wild-type DHFR protein is employed, itis preferable to select a host cell that is deficient in DHFR, thuspermitting the use of the DHFR coding sequence as a marker forsuccessful transfection in selective medium that lacks hypoxanthine,glycine, and thymidine. An appropriate host cell in this case is theChinese hamster ovary (CHO) cell line deficient in DHFR activity,prepared and propagated as described by Urlaub and Chasin, Proc. Natl.Acad. Sci. (USA) 77, 4216 (1980).

On the other hand, if DHFR protein with low binding affinity for MTX isused as the controlling sequence, it is not necessary to useDHFR-deficient cells. Because the mutant DHFR is resistant tomethotrexate, MTX-containing media can be used as a means of selectionprovided that the host cells are themselves methotrexate sensitive. Mosteukaryotic cells that are capable of absorbing MTX appear to bemethotrexate sensitive. One such useful cell line is a CHO line, CHO-K1(ATCC No. CCL 61).

b. Typical Methodology Employable

Construction of suitable vectors containing the desired coding andcontrol sequences employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and religated in theform desired to prepare the plasmids required.

If blunt ends are required, the preparation may be treated for 15minutes at 15° C. with 10 units of Polymerase I (Klenow),phenol-chloroform extracted, and ethanol precipitated.

Size separation of the cleaved fragments may be performed using 6percent polyacrylamide gel described by Goeddel et al., Nucleic AcidsRes. 8, 4057 (1980).

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are typically used to transform E. coli K12 strain 294(ATCC 31,446) or other suitable E. coli strains, and successfultransformants selected by ampicillin or tetracycline resistance whereappropriate. Plasmids from the transformants are prepared and analyzedby restriction mapping and/or DNA sequencing by the method of Messing etal., Nucleic Acids Res. 9, 309 (1981) or by the method of Maxam et al.,Methods of Enzymology 65, 499 (1980).

After introduction of the DNA into the mammalian cell host and selectionin medium for stable transfectants, amplification of DHFR-protein-codingsequences is effected by growing host cell cultures in the presence ofapproximately 20,000-500,000 nM concentrations of methotrexate, acompetitive inhibitor of DHFR activity. The effective range ofconcentration is highly dependent, of course, upon the nature of theDHFR gene and the characteristics of the host. Clearly, generallydefined upper and lower limits cannot be ascertained. Suitableconcentrations of other folic acid analogs or other compounds thatinhibit DHFR could also be used. MTX itself is, however, convenient,readily available, and effective.

Other techniques employable are described in the Examples.

c. VEGF Receptor-Immunoglobulin Chimeras (Immunoadhesins)

Immunoglobulins and certain variants thereof are known and may have beenprepared in recombinant cell culture. For example, see U.S. Pat. No.4,745,055; EP 256,654; Faulkner et al., Nature 298:286 (1982), EP120,694, EP 125,023, Morrison, J. Immunol. 123:793 (1979); Kohler etal., Proc. Natl. Acad. Sci. USA 77:2197 (1980); Raso et al., Cancer Res.41:2073 (1981); Morrison, Ann. Rev. Immunol. 2:239 (1984); Morrison,Science 229:1202 (1985); Morrison et al., Proc. Natl. Acad. Sci. USA81:6851 (1984); EP 255,694, EP 266,663; and WO 88/03559. Reassortedimmunoglobulin chains are also known. See, for example, U.S. Pat. No.4,444,878; WO 88/03565; and EP 68,763 and references cited therein.

Chimeras constructed from a protein receptor sequence linked to anappropriate immunoglobulin constant domain sequence (immunoadhesins) areknown in the art. Immunoadhesins reported in the literature includefusions of the T cell receptor (Gascoigne et al. Proc. Natl. Acad. Sci.USA 84:2936-2940 [1987]), CD4 (Capon et al., Nature 337:525-531 [1989]),L-selectin (homing receptor) (Watson et al., J. Cell. Biol.110:2221-2229 [1990]), CD44 (Aruffo et al., Cell 61:1303-1313 [1990]),CD28 and B7 (Linsley et al., J. Exp. Med. 173:721-730 [1991]), CTLA-4(Linsley et al., J. Exp. Med. 174:561-569 [1991]), CD22 (Stamenkovic etal., Cell 66:1133-1144 [1991]), TNF receptor (Ashkenazi et at., Proc.Natl. Acad. Sci. USA 88:10535-10539 [1991]) and IgE receptor alpha(Ridgway et al., J. Cell. Biol. 115:abstr. 1448 [1991]).

The simplest and most straightforward immunoadhesin design combined thebinding region(s) of an “adhesin” protein with the Fc regions of animmunoglobulin heavy chain. Ordinarily, when preparing the chimeric VEGFreceptors of the present invention having immunoglobulin sequences,nucleic acid encoding the Ig-like domains of the VEGF receptor(s) willbe fused C-terminally to nucleic acid encoding the N-terminus of animmunoglobulin constant domain sequence, however, N-terminal fusions arealso possible.

Typically, in such fusions, the encoded chimeric polypeptide will retainat least functionally active hinge, CH2 and CH3 domains of the constantregion of an immunoglobulin heavy chain. Fusions are also made to theC-terminus of the Fc porion of a constant domain, or immediatelyN-terminal to the CH1 of the heavy chain or the corresponding region ofthe light chain.

The precise site at which the fusion is made is not critical; particularsites are well known and may be selected in order to optimize thebiological activity, secretion or binding characteristics of the VEGFreceptor/immunoglobulin chimera.

In some embodiments, the VEGF receptor/Ig chimeras of the presentinvention may be assembled as monomers, or hetero- or homo-multimers;and particularly as dimers and trimers, essentially as illustrated in WO91/08298.

In a preferred embodiment, the VEGF receptor Ig-like domains of interestare fused to the N-terminus of the Fc domain of immunoglobulin G₁(IgG-1). It is possible to fuse the entire heavy chain constant regionto the VEGF receptor Ig-like domains of interest. However, morepreferably, a sequence beginning in the hinge region just upstream ofthe papain cleavage site which defines Fc chemically, or analogous sitesof other immunoglobulins are used in the fusion. In a particularlypreferred embodiment, the Ig-like domains of the VEGF receptor ofinterest are fused to (a) the hinge region and CH₂ and CH₃ or (b) theCH₁, hinge, CH2 and CH3 domains, of an IgG-1, IgG-2 or IgG-3 heavychain. The precise site at which the fusion is made is not critical, andthe optimal site can be determined by routine experimentation.

In some embodiments, the Ig-like domains VEGF receptor/immunoglobulinchimeras of the present invention are assembled as multimers, andparticularly as homo-dimers or -tetramers. Generally, these assembledimmunoglobulins will have known unit structures. A basic four chainstructural unit is the form in which IgG, IgD, and IgE exist. A fourchain unit is repeated in the higher molecular weight immunoglobulins;IgM generally exists as a pentamer of four basic units held together bydisulfide bonds. IgA globulin, and occasionally IgG globulin, may alsoexist in multimeric form in serum.

Ig-like domain sequences from the VEGF receptors can also be insertedbetween immunoglobulin heavy and light chain sequences, such that animmunoglobulin comprising a chimeric heavy chain is obtained. In thisembodiment, the VEGF receptor Ig-like sequences are fused to the 3′ endof an immunoglobulin heavy chain in each are of the immunoglobulin,either between the hinge and the CH2 domain, or between the CH2 and CH3domains. Similar constructs have been reported by Hoogenboom, et al.Mol. Immunol. 28:1027-1037 (1991).

Although the presence of an immunoglobulin light chain is not requiredin the immunoadhesins of the present invention, an immunoglobulin lightchain might be present either covalently associated to a VEGF receptorIg-like domain-immunoglobulin heavy chain fusion polypeptide, ordirectly fused to the VEGF receptor Ig-like domains. In the former case,DNA encoding an immunoglobulin light chain is typically coexpressed withthe VEGF receptor Ig-like domain-immunoglobulin heavy chain chimericprotein. Upon secretion, the hybrid heavy chain and light chain will becovalently associated to provide an immunoglobulin-like structurecomprising two disulfide-linked immunoglobulin heavy chain-light chainpairs. Methods suitable for the preparation of such structures are, forexample, disclosed in U.S. Pat. No. 4,816,567, issued 28 Mar. 1989.

In a preferred embodiment, the immunoglobulin sequences used in theconstruction of the immunoadhesins of the present invention are from anIgG immunoglobulin heavy chain domain. For human immunoadhesins, the useof human IgG1 and IgG3 immunoglobulin sequences is preferred. A majoradvantage of using the IgG1 is that IgG1 immunoadhesins can be purifiedefficiently on immobilized protein A. However, other structural andfunctional properties should be taken into account when choosing the Igfusion partner for a particular immunoadhesin construction. For example,the IgG3 hinge is longer and more flexible, so that it can accommodatelarger “adhesin” domains that may not fold or function properly whenfused to IgG1. Another consideration may be valency; IgG immunoadhesinsare bivalent homodimers, whereas Ig subtypes like IgA and IgM may giverise to dimeric or pentameric structures, respectively, of the basic Ighomodimer unit. For VEGF receptor Ig-like domain/immunoglobulin chimerasdesigned for in vivo applications, the pharmacokinetic properties andthe effector functions specified by the Fc region are important as well.Although IgG1, IgG2 and IgG4 all have in viva half-lives of 21 days,their relative potencies at activating the complement system aredifferent. Moreover, various immunoglobulins possess varying numbers ofallotypic isotypes.

The general methods suitable for the construction and expression ofimmunoadhesins are the same as those described herein above with regardto (native or variant) Ig-like domains of the various VEGF receptors.Chimeric immunoadhesins of the present invention are most convenientlyconstructed by fusing the cDNA sequence encoding the VEGF receptorIg-like domain(s) of interest in-frame to an Ig cDNA sequence. However,fusion to genomic Ig fragments can also be used (see, e.g., Gascoigne atal., supra). The latter type of fusion requires the presence of Igregulatory sequences for expression. cDNAs encoding IgG heavy chainconstant regions can be isolated based on published sequences from cDNAlibraries derived from spleen or peripheral blood lymphocytes, byhybridization or by polymerase chain reaction (PCR) techniques. ThecDNAs encoding the “adhesin” derived from the VEGF receptor Ig-likedomain(s) and the Ig parts of the chimera are inserted in tandem into aplasmid vector that directs efficient expression in the chosen hostcells. The exact junction can be created by removing the extra sequencesbetween the designed junction codons using oligonucleotide-directeddeletional mutagenesis (Zoller and Smith, Nucl. Acids Res. 10:6487(1982)). Synthetic oligonucleotides can be used, in which each half iscomplementary to the sequence on either side of the desired junction.Alternatively, PCR techniques can be used to join the two parts of themolecule in-frame with an appropriate vector.

The chimeric immunoadhesins of the present invention can be purified byvarious well known methods including affinity chromatography on proteinA or G, thiophilic gel chromatography (Hutchens at al., Anal. Biochem.159:217-226 [1986]) and immobilized metal chelate chromatography(Al-Mashikhi at al., J. Dairy Sol. 71:1756-1763 [1988]).

d. Therapeutic Uses and Formulations

For therapeutic applications, the chimeric VEGF receptor proteins of thepresent invention are administered to a mammal, preferably a human, in apharmaceutically acceptable dosage form, including those that may beadministered to a human intervenously as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intra-cerebrospinal, subcutaneous, intra-arterial, intrasynovial,intrathecal, oral, topical, or inhalation routes. The chimeric VEGFreceptor proteins of the present invention are also suitablyadministered by intratumoral, peritumoral, intralesional or perilesionalroutes, to exert local as well as systemic effects. The intraperitonealroute is expected to be particularly useful, for example, in thetreatment of ovarian tumors.

Such dosage forms encompass pharmaceutically acceptable carriers thatare inherently nontoxic and nontherapeutic. Examples of such carriersinclude ion exchangers, alumina, aluminum stearate, lecithin, serumproteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts, orelectrolytes such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, and polyethylene glycol. Carriers for topical or gel-basedforms of chimeric protein include polysaccharides such as sodiumcarboxymethylcellulose or methylcellulose, polyvinylpyrrolidone,polyacrylates, polyoxyethylene-polyoxypropylene-block polymers,polyethylene glycol and wood wax alcohols. For all administrations,conventional depot forms are suitably used. Such forms include, forexample, microcapsules, nano-capsules, liposomes, plasters, inhalationforms, nose sprays, sublingual tablets, and sustained releasepreparations. For examples of sustained release compositions, see U.S.Pat. No. 3,773,919, EP 58,481A, U.S. Pat. No. 3,887,699, EP 158,277A,Canadian Patent No. 1176565, U. Sidman et al., Biopolymers 22:547 (1983)and R. Langer at al., Chem. Tech. 12:98 (1982). The chimeric proteinwill usually be formulated in such vehicles at a concentration of about0.1 mg/ml to 100 mg/ml.

Optionally other ingredients may be added to pharmaceutical formulationsof the chimeric VEGF receptor proteins of the present invention such asantioxidants, e.g., ascorbic acid; low molecular weight (less than aboutten residues) polypeptides, e.g., polyarginine or tripeptides; proteins,such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone; amino acids, such as glycine, glutamicacid, aspartic acid, or arginine; monosaccharides, disaccharides, andother carbohydrates including cellulose or its derivatives, glucose,mannose, or dextrins; chelating agents such as EDTA; and sugar alcoholssuch as mannitol or sorbitol.

The chimeric VEGF receptor protein formulation to be used fortherapeutic administration must be sterile. Sterility is readilyaccomplished by filtration through sterile filtration membranes (e.g.,0.2 micron membranes). The chimeric VEGP receptor protein ordinarilywill be stored in lyophilized form or as an aqueous solution if it ishighly stable to thermal and oxidative denaturation. The pH of thechimeric VEGF receptor protein preparations typically will be about from6 to 8, although higher or lower pH values may also be appropriate incertain instances.

For the prevention or treatment of disease, the appropriate dosage ofchimeric VEGF receptor protein will depend upon the type of disease tobe treated, the severity and course of the disease, whether the chimericVEGF receptor proteins are administered for preventative or therapeuticpurposes, previous therapy, the patient's clinical history and responseto the chimeric VEGF receptor protein and the discretion of theattending physician. The chimeric VEGF receptor protein is suitableadministered to the patient at one time or over a series of treatments.For purposes herein, the “therapeutically effective amount” of achimeric VEGF receptor protein is an amount that is effective to eitherprevent, lessen the worsening of, alleviate, or cure the treatedcondition, in particular that amount which is sufficient to reduce orinhibit the proliferation of vascular endothelium in vivo.

The chimeric VEGF receptor proteins of the present invention are usefulin the treatment of various neoplastic and non-neoplastic diseases anddisorders. Neoplasms and related conditions that are amenable totreatment include carcinomas of the breast, lung, esophagus, gastricanatomy, colon, rectum, liver, ovary, cervix, endometrium, thecomas,arrhenoblastomas, endometrial hyperplasia, endometriosis, fibrosarcomas,choriocarcinoma, head and neck cancer, nasopharyngeal carcinoma,laryngeal carcinoma, hepatoblastoma, Karposi's sarcoma, melanoma, skincarcinomas, hemangioma, cavernous hemangioma, hemangioblastoma, pancreascarcinoma, retinoblastoma, astrocytoma, glioblastoma, Schwannoma,oligodendroglioma, medulloblastoma, neuroblastomas, rhabdomyosarcoma,osteogenic sarcoma, leiomyosarcomas, urinary tract carcinomas, thyroidcarcinomas, Wilm's tumor, renal cell carcinoma, prostate carcinoma,abnormal vascular proliferation associated with phakomatoses, edema(such as associated with brain tumors), and Meigs' syndrome.

Non-neoplastic conditions that are amenable to treatment includerheumatoid arthritis, psoriasis, atherosclerosis, diabetic and otherretinopathies, retrolentral fibroplasia, neovascular glaucoma,age-related macular degeneration, thyroid hyperplasias (includinggrave's disease), corneal and other tissue transplantation, chronicinflammation, lung inflammation, nephrotic syndrome, preclampasia,ascites, pericardial effusion (such as associated with pericarditis) andpleural effusion.

The following examples are intended merely to illustrate the best modenow known for practicing the invention but the invention is not to beconsidered as limited to the details of such examples.

Example 1 Construction and Analysis of the flt-1 ExtracellularDomain/IgG F_(c) Chimera (flt-1/IgG) and Deletion Constructs Thereof

An expression construct consisting of the native flt-1 extracellularligand-binding region, having seven Ig-like domains, fused to the F_(c)portion of human IgG₁ was constructed essentially as described by Parket al., J. Biol. Chem. 269:25646-25654 (1994) which is expresslyincorporated herein by reference. Specifically, the extracellularligand-binding region of the flt-1 receptor was cloned by the polymerasechain reaction using Pfu polymerase. Human placental cDNA served as thetemplate. Primers encompassed the entire extracellular ligand-bindingregion of the cDNA encoding the flt-1 extracellular ligand-bindingregion, including signal peptides. Shibuya et al., Oncogene 5:519-524(1990) and deVries et al., Science 255:989-991 (1992). The cDNA for theflt-1 extracellular ligand-binding region was cloned in two pieces tofacilitate sequencing. Two sets of primers (shown below) were used, andthe resulting band (approximately 1 kilobase in size) was digested withthe appropriate enzymes and subcloned into pBluescript II or pSL301. ThecDNA produced encoded the first 758 amino acids of the flt-1 receptor.Full-length flt-1 extracellular ligand-binding region cDNA was createdby ligating the two flt-1 polymerase chain reaction clones at a uniquenatural Mun I restriction site.

flt-1 primer set #1:

5′ TCTAGAGAATTCCATGGTCAGCTACTGGGACACC 3′ 5′ CCAGGTCATTTGAACTCTCGTGTTC 3′flt-1 primer set #2:

5′ TACTTAGAGGCCATACTCTTGTCCT 3′ 5′ GGATCCTTCGAAATTAGACTTGTCCGAGGTTC 3′These primers changed amino acid 757 of the flt-1 extracellularligand-binding region to phenylalanine and introduced a Bst BI site atthe 3′ end thereof. A Bst BI mutation which eliminated any linkersequences was introduced at the 5′ end of CH₂CH₃, an IgGy1 heavy chaincDNA clone. Capon, at al., Nature 337:525-531 (1989). The flt-1extracellular ligand-binding region sequences were then fused to thecoding sequences for amino acids 216-443 of this IgGy1 heavy chain clonevia the unique Bst BI site at the 3′ end of the flt-1 extracellularligand-binding region coding region. This construct was then subclonedinto the plasmid pHEBO23 for expression in CEN4 cells as described byPark at al., Mol. Biol. Cell. 4:1317-1326 (1993). The authenticity ofthe clone was verified by DNA sequencing. This resulted in thesuccessful construction of the chimera “flt-1/IgG” wherein the nativeextracellular ligand-binding region of the flt-1 receptor is fused tothe F_(c) portion of human IgG.

The amino acid sequences of the extracellular ligand-binding region ofthe fit-1, KDR and FLT4 receptors were then aligned using the sequenceanalysis program “align” and the boundaries of each of the seven Ig-likedomains present in the extracellular ligand-binding regions of theflt-1, KDR and FLT4 receptors were determined by structural and sequenceconsiderations. FIG. 1 presents the alignment of the extracellularligand-binding regions of the flt-1, KDR and FLT4 receptors.

Once the boundaries of the seven Ig-like domains in the extracellularligand-binding regions of the flt-1, KDR and FLT4 receptors were definedas shown in FIG. 1, the flt-1/IgG construct prepared above was utilizedas a template to systematically delete each of the seven individualIg-like domains of the flt-1 extracellular ligand-binding region byemploying the “loop-out” mutagenesis technique previously described byUrfer et al., EMBO J. 14:2795-2805 (1995). See also Kunkle, Proc. Natl.Acad. Sci. USA 82:488-492 (1985). Specifically, by utilizingoligonucleotide-directed mutagenesis, oligonucleotides were designed to“loop-out” a single Ig-like domain from the flt-1/IgG construct whilealso creating unique restriction sites at the boundaries of each of theseven Ig-like domains to be used for inserting other Ig-like domainsobtained from other . VEGF receptor extracellular ligand-binding regionsfor the purpose of creating other chimeric VEGF receptor molecules (seebelow). FIG. 2 presents the oligonucleotides used in generating Ig-likedomain deletions from the flt-1/IgG construct and the restriction sitescreated as such. These experiments resulted in obtaining sevenadditional flt-1/IgG constructs, each having one of the seven differentIg-like domains deleted out.

To create the flt-1 Ig-like domain 1 deletion, the oligonucleotide5′-AAAA TTAAAAGATCCAGATCTGACTATCTATATATTTATTAGTGATACCGGTAG ACCTTTT-3′was used to “loop-out” amino acids 36-123 and to introduce a Bgl II siteand an Age I site at the 5′- and 3′-ends of the domain 1 deletion,respectively (see FIG. 2). Creation of the Bgl II site changed aminoacid E33 to D.

The oligonucleotide used to create the flt-1 Ig-like domain 2 deletionwas 5′-GAAGGAAACAGAAGGCGCCATCTATATATTTATTCGAGGTACCAATA CAATCATAG-3′,effectively removing amino acids S129 through H223. The creation of KasI and Kpn I restriction sites caused amino acid changes S122 to G andQ225 to G to occur, respectively.

Deletion of the third Ig-like domain removed amino acids N227 throughS325 using the oligonucleotide 5′-CAAACTATCTCACACATAGATCTACCGTGCATATATATGATACCGGTTTCATCACTGTGAAAC-3′. Amino acids Q225, K331 andA332 were changed to S, T and G, respectively to accommodate for theinsertion of Bgl II and Age I restriction sites.

The oligonucleotide 5′-GTTAACACCTCAGTGCACGTGTATGATGTCAATGTGAAACCCCAGATCTACGAAAAGGCCGTGTC-3′ was used to loop-out amino acids K331though I423 (fit-1 Ig-like domain 4 deletion). The amino acid changeresulting from the generation of a Bbr PI restriction site was I328 toV. Constituting a Bgl II restriction site at the 3′ end of Ig-likedomain 4 did not alter any amino acids.

Deleting Ig-like domain 5 amino acids .K427 through S549 was achievedutilizing the oligonucleotide 5′-AAACCTCACTGCCACGCTAGCTGTCAATGTGTTTTATATCACAGATCTGCCAAATGGGTTTCAT-3′. Devising an Nhe I restrictionsite at the 5′ end mutated I423 to A; amino acid V555 was substituted byL during the insertion of the Bgl II site in the 3′ end.

To generate the Ig-like domain 6 deletion mutant, the oligonucleotide5′-G TGGGAAGAAACATAAGCTTTGTATACATTACAATCAGATCTCAGGAAGC ACCATAC-3′excised the amino acids T553 through E652. Generating the Bst 1707Irestriction site at the 5′ end changed amino acids Y551 and I552 to Vand Y, respectively; amino acid D657 was substituted by S during theformation of the Bgl II restriction site at the 3′ end.

The last flt-1 Ig-like domain to be deleted, domain 7, removed aminoacids Q658 through Y745 while adding restriction site Bsi WI and Kpn I5′ and 3′, respectively. The oligonucleotide, 5′-CCAGAAGAAAGAAATTACCTACGAGATCTCACTGTTCAAGGTACCTCGGACAAGTCTAAT-3′, did cause an aminoacid substitution at I655 into V.

Following construction of the flt-1/IgG construct and the Ig-like domaindeletion constructs based on flt-1/IgG, the constructs wereindependently transformed into E. coli strain XL-1 Blue using techniqueswell known in the art. Following transformation of the constructs intoE. coli strain XL-1 Blue, colonies were tested via restriction digestionfor the presence of the newly created restriction sites shown in FIG. 2and subsequently the entire coding region of each construct wassequenced using the Sequenase version 2.0 kit (US Biochemical Corp.).Double-stranded DNA for each selected clone was prepared using theQIAGEN DNA purification kit (Qiagen Inc.) and was used for transfectioninto CEN4 cells.

Plasmid DNA coding for the native flt-1/IgG protein or the flt-1/IgGdomain deletions was introduced into CEN4 cells by calcium phosphateprecipitation (Current protocols in Molecular Biology). CEN4 cells are aderivative of the human embryonic kidney 293 cell line that expressesthe Epstein-Barr virus nuclear antigen-1, required for episomalreplication of the pHEBO23 vector upon which the flt-1/IgG construct isbased. Su at al., Proc. Natl. Acad. Sci. USA 88:10870-10874 (1991). Tenμg of plasmid DNA was used for transfection of a single 80% confluent 10mm cell culture dish. Forty-eight hours post-transfection, the mediacontaining the soluble chimeric VEGF receptors was collected and theconcentration of protein produced was determined by ELISA assaysdesigned to detect the F_(c) portion of the chimeric protein.

Example 2 Binding Assays for Detecting Binding to the VEGF Ligand

Binding assays with the soluble chimeric VEGF receptors generated inExample 1 above were performed essentially as described by Park at al.,J. Biol. Chem. 269:25646-25654 (1994). Specifically, binding assays wereperformed in ninety-six-well breakaway immunoabsorbent assay plates(Nunc) coated overnight at 4° C. with 2 μg/ml affinity-purified goatanti-human F_(c) IgG (Organon-Teknika) in 50 mM Na₂CO₃, pH 9.6. Plateswere blocked for 1 hr with 10% fetal bovine serum in PBS (buffer B).After removal of the blocking buffer, 100 μl of a binding cocktail wasadded to each well. Binding cocktails consisted of a given amount of anflt-1/IgG chimeric protein, ¹²⁵I-VEGF₁₆₅ (<9000 cpm/well), plus or minus50 ng of unlabeled VEGF competitor where indicated, all within buffer Bfor a final volume of 100 μl; the cocktails were assembled and allowedto equilibrate overnight at 4° C. VEGF₁₆₅ was iodinated by thechloramine T method as previously described by Keyt et al., J. Biol.Chem. 271:5638-5646 (1996). The specific activity of the iodinated VEGFwas 5.69×10⁷ cpm/microgram. Incubation in the coated wells proceeded for4 hrs at room temperature, followed by 4 washes with buffer B. Bindingwas determined by counting individual wells in a gamma counter. Data wasanalyzed using a 4-parameter non-linear curve fitting program(Kalidagraph, Abelbeck Software).

The results of the binding assays employing the intact flt-1/IG chimericprotein and the seven flt-1/IgG Ig-like deletion chimeric proteins arepresented in FIG. 3. As shown in FIG. 3, of all of the chimeric proteinstested, only the chimeric protein lacking the Ig-like domain 2 wasunable to bind the VEGF ligand specifically. All of the other sixflt-1/IgG deletion chimeras tested, as well as the intact flt-1/IgGchimera, retained the ability to bind the VEGF ligand specifically.These results demonstrate that the Ig-like domain 2 of the flt-1extracellular ligand-binding region is required for specific binding tothe VEGF ligand.

These results lead to the cloning, expression and testing of variousother fit-1/IgG deletion chimeric constructs. Specific Ig-likedomain/IgG constructs were created by amplifying the specific Ig-likedomains desired, using PCR primers containing restrictions sites (Cla Iand Bst BI, 5′ and 3′, respectively) which provided the in-frame sitesto clone into the 5′ end of the IgG1 heavy chain cDNA plasmid (see Caponet al., Nature 337:525-531 (1989)). This resulted in the construction ofan flt-1/IgG deletion construct having only Ig-like domains 1 and 2 ofthe flt-1 extracellular ligand-binding region fused to the F_(c) of IgG[flt(1,2)]. For the construction of flt(1,2), amino acids M1 throughQ224 were amplified using oligonucleotides5′-CAGGTCAATCATCGATGGTCAGCTACTGGGACACC-3′ (Flt.sp.Cla I) and5′-GGTCAACTATTTCGAATTGTCGATGTGTGAGATAG-3′(Flt.2C.Bst BI).

Other flt-1/IgG deletion chimeras were also similarly prepared andcontained the combination of Ig-like domain 2 only [flt(2)], Ig-likedomains 2 and 3 only [flt(2,3)] and Ig-like domains 1, 2 and 3 only[flt(1,2,3)]. The same two oligonucleotides used to crease flt(1,2) werealso used to create flt(2) from a construct lacking Ig-like domain 1.Flt(2,3) was generated by amplifying a construct lacking Ig-like domain1 with the Flt.sp.Cla I oligonucleotide and another oligonucleotide5′-GGTCAACTATTTCGAATATATGCACTGAGGTGTTAAC-3′ (Flt.3C.Bst BI) whichincludes the coding sequence through I328. Amplifying flt-1 Ig-likedomains 1 through 3 was accomplished using primers Flt.sp.Cla I andFit.3C.Bst BI on a construct having all three Ig-like domains. Theentire domain-IgG coding sequence was then subcloned into pHEBO23 at theCla I and Not 1 sites.

All of these flt-1/IgG chimera constructs were cloned, expressed andtested for their ability to specifically bind to VEGF as described abovein Examples 1 and 2. As with the other flt-1/IgG constructs, all ofthese flt-1/IgG deletion constructs were sequenced, transfected intoCEN4 cells and the expressed protein quantitated by F_(c) ELISA. Theresults of the VEGF binding assays with the flt-1/IgG domain deletionchimeras is presented in FIG. 4.

The results presented in FIG. 4 demonstrate that flt-1 Ig-like domain 2by itself is insufficient to allow binding of the VEGF ligand. Ig-likedomain 1 in combination with domain 2 was also not sufficient to allowbinding of the VEGF ligand. A small amount of VEGF-binding could bedetected when Ig-like domains 2 and 3 were present in combination, butthe extent and affinity of this binding needs to be further analyzed. Incontrast, however, the ability to bind the VEGF ligand was completelyrestored when Ig-like domains 1, 2 and 3 were all three present incombination. These results, therefore, demonstrate that the combinationof flt-1 Ig-like domains 1, 2 and 3 is sufficient for VEGF binding.

Next, increasing amounts of unlabeled VEGF ligand was used to titrate¹²⁵I-VEGF₁₆₅ binding to 1ng of immunoreactive flt-1/IgG or flt(1,2,3).Binding assays were performed essentially as described above. Theresults from these experiments are presented in FIG. 5. Using the 4 Plogistic curve fit: [(m1−m4)/(1+(m0/m3) ̂m2)]+m4, the value of m3 equalsthe concentration resulting in 50% inhibition (IC₅₀). This occurs at thepoint of inflection of the curve. As is shown in FIG. 5, the IC₅₀ forthe intact flt-1/IgG chimeric protein and flt(1,2,3) deletion chimera issimilar at 1.89 ng/ml and 1.34 ng/ml, respectively. Thus, the flt(1,2,3)deletion chimera behaves in a similar fashion to the intact flt-1/IgGchimeric protein with respect to binding to the VEGF ligand.

Example 3 Binding Assays for Detecting Binding by “Swap” Chimeras to theVEGF Ligand

As shown in FIG. 1, the boundaries for each of the seven Ig-like domainspresent within the extracellular ligand-binding regions of the flt-1,KDR and FLT4 receptors were determined. Based on this information,various “swap” chimeras were prepared where one or more of the Ig-likedomains from the flt-1/IgG construct were replaced with the same Ig-likedomains from either the KDR or FLT4 receptor. In order to constructthese “swap” chimeras, the desired domain fragment from either the KDRor FLT4 receptor was amplified using PCR primers which contained thesame flanking restriction sites in frame as were created during theconstruction of the intact flt-1/IgG construct described above. Cleavingboth the intact flt-1/IgG construct and the PCR fragment obtained fromthe amplification of the KDR or FLT4 receptor DNA with the restrictionenzymes and subsequent ligation of the resulting fragments yieldedconstructs coding for the desired “swap” chimeras. All chimericconstructs produced were sequenced to confirm their authenticity.

In one experiment, the Ig-like domain 2 of either the KDR or FLT4receptor was “swapped” for the Ig-like domain 2 of the flt-1/IgGconstruct to produce “swap” chimeras having flt-1 Ig-like domains 1 and3-7 in combination with Ig-like domain 2 from either KDR (flt.K2) orFLT4 (fitF4.2). As before, both “swap” constructs were sequenced priorto transfection into and expression in CEN4 cells and the “swap”chimeras produced by the CEN4 cells were subjected to F_(c) ELISA. Thechimeric proteins were then tested as described above for their abilityto specifically bind to ¹²⁵I-VEGF₁₆₅. The results are presented in FIG.6.

The results presented in FIG. 6 demonstrate that replacing the flt-1Ig-like domain 2 with the Ig-like domain 2 of the KDR receptor functionsto re-establish the ability to specifically bind to the VEGF ligandwhereas the presence of FLT4 Ig-like domain 2 did not re-establish theability to specifically bind to the VEGF ligand. Since it is known thatnative FLT4 receptor does not bind to the VEGF ligand and since the KDRreceptor does interact with this ligand, these results demonstrate thatIg-like domain 2 is the primary domain responsible for VEGF binding.Expectedly, the native flt-1/IgG chimera specifically bound to the VEGFligand whereas the flt-1/IgG chimera lacking an Ig-like domain 2 did notspecifically bind to the VEGF ligand.

Next, experiments were performed to determine if the specificity forbinding to the VEGF ligand resides in the Ig-like domain 2 of the flt-1and KDR receptors. Specifically, it is well known that placenta growthfactor (PLGF) is capable of binding to the extracellular ligand-bindingregion of the flt-1 receptor but does not bind to the extracellularligand-binding regions of either the KDR or FLT4 receptors. Thus,binding of PLGF can compete with binding of the VEGF ligand to the flt-1receptor.

Based on this information, a competition against VEGF binding wasperformed using a series of “swap” mutants that consisted of theflt-1/IgG chimeric protein wherein various Ig-like domains thereof werereplaced with the same Ig-like domains from the KDR receptor.Specifically, “swap” chimeras were constructed as described abovewherein either Ig-like domain 1, 2, 3, 5 or 7 of the flt-1/IgG chimerawas replaced by the corresponding Ig-like domain from the KDR receptor.Competition binding assays were performed as described above whereincompetitors consisted of 50 ng of unlabeled VEGF or 50 ng of unlabeledPLGF. The results of these competition binding assays are presented inFIG. 7.

The results presented in FIG. 7 demonstrate that only when the Ig-likedomain 2 of the flt-1 receptor is replaced with the Ig-like domain 2 ofthe KDR receptor is the VEGF interaction more like wild type KDR thanwild type flt-1. Each of the other “swap” chimeras constructed behavedsimilar to the wild type flt-1 receptor. Moreover, when the Ig-likedomain 2 of the flt-1/IgG chimeric protein was replaced by the Ig-likedomain 2 of the FLT4 receptor, the resulting chimeric protein exhibitedthe binding specificity of the intact FLT4 receptor (data not shown).These results demonstrate, therefore, that the Ig-like domain 2 of theflt-1 and KDR receptors is the major determinant of ligand specificity.

Next, an expression construct encoding the entire human FLT4 receptor,including the extracellular domain, transmembrane region andintracellular tyrosine kinase domain (Lee at al., Proc. Natl. Acad. Sc.USA 93:1988-1992 (1996)) was used to create various other chimericreceptors. The construct encoding the entire FLT4 receptor was thensubjected to oligo-directed mutagenesis as described above to createin-frame restriction sites located at the beginning of the Ig-likedomain 1 of the FLT4 extracellular ligand-binding region (Afl II), theend of domain 1/beginning of domain 2 (Nhe I), the end of domain2/beginning of domain 3 (Bsi WI), and the end of domain 3 (Mlu I). Thefollowing flt-1 Ig-like domain combinations were then amplifiedessentially as described above using PCR primers that possessed the samein-frame restriction sites: domain 2 alone and domains 1-3 alone.Cloning the flt-1 PCR products into the mutagenized FLT4 encodingconstruct resulted in flt-1/FLT4 chimeric receptor constructs.Specifically, constructs were prepared which possessed the entire FLT4receptor sequences except that the FLT4 Ig-like domains 1-3 werereplaced with the Ig-like domains 1-3 of the flt-1 receptor (constructflt-1(1,2,3)/FLT4) or that the FLT4 Ig-like domain 2 was replaced withthe Ig-like domain 2 of the flt-1 receptor (construct flt-1(2)/FLT4).For flt-1(1,2,3)/FLT4, FLT4 sequence encoding amino acids N33 throughE324 was replaced by flt-1 sequences encoding S35 through S325. Creationof the cloning sites resulted in a change of I325 of FLT4 to R. Forflt-1(2)/FLT4, FLT4 sequence encoding S128 through I224 was replaced byflt-1 sequence encoding I124 through R224. This also changes FLT4 aminoacids N33 and I326 to S and R, respectively and added T36. Sequencingconfirmed the authenticity of these chimeras. FIGS. 8, 9 and 10 show theentire amino acid sequences of the intact FLT4 receptor, the entireamino acid sequence of the chimeric receptor encoded by theflt-1(1,2,3)/FLT4 construct and the entire amino acid sequence of thechimeric receptor encoded by the flt-1(2)/FLT4 construct, respectively.

After preparation of these expression constructs, 293 cells weretransfected with the constructs via DEAE-Dextran andtransiently-expressing cells were analyzed for the ability to bind tothe VEGF ligand. To detect binding of the VEGF ligand, a saturationbinding assay was performed on transiently-expressing 293 cellsexpressing the intact FLT4 receptor, the flt-1 domain 2/FLT4 chimericreceptor, the flt-1 domains 1-3/FLT4 chimeric receptor, or the intactflt-1 receptor. Specifically, 2.5×10⁵ cells were incubated withincreasing amounts of ¹²⁵I-VEGF (specific activity of 56.9×10⁵ cpm/μg)in a final volume of 0.2 mls of buffer C (50/50 media with 0.1% BSA and25 mM HEPES pH 7.3) for 4 hrs at 4° C. with slight agitation. The cellmixture was then layered over a 0.75 ml cushion of 30% sucrose,centrifuged for 10 minutes at maximum speed, and the pellet wasrecovered and counted in a gamma counter. Because 293 cells possess someflt-1-like VEGF binding, non-transfected cells were also used and thebackground counts were subtracted out from the counts recovered for thetransfected cells. The amount of counts added and the recovered countsbound were then subjected to scatchard analysis.

The results of these experiments demonstrated that, as expected, thecells expressing the intact FLT4 receptor did not specifically bind theVEGF ligand. However, cells expressing the flt-1 domain 2/FLT4 chimericreceptor or the flt-1 domains 1-3/FLT4 chimeric receptor didspecifically bind the VEGF ligand specifically and tightly. The Kds areapproximately 10.2 pM+/−1.1 pM and 10.4 pM+/−3.4 pM, respectively, forthe flt-1 domain 2/FLT4 chimeric receptor and the flt-1 domains 1-3/FLT4chimeric receptor. These values are near the range reported for theintact full-length fit-1 receptor.

Experiments were also performed to measure the amount of tyrosinephosphorylation in 293 cells transiently expressing these chimericreceptors 60-72 hours post-transfection. Tyrosine phosphorylation assayswere performed essentially as described in Park at al., J. Biol. Chem.269:25646-25654 (1994). The transiently expressing 293 cells weredeprived of serum 16-18 hrs prior to stimulation by a given factor.Cells were stimulated with FLT4 ligand (VH1.4.5; VEGF-C/VRP) at aconcentration of 400 ng/ml, 50 ng/ml VEGF, or 0.5 nM PLGF for 15 minutesat 37° C. Following removal of the stimulation media, the cells weretwice washed with ice-cold PBS and then lysed in 1 ml lysis buffer. Thelysate was cleared of cellular debris and the receptors wereimmunoprecipitated using JTL.1, a polyclonal antibody directed againstthe extracellular domain of the FLT4 receptor (see Lee et. al., Proc.Natl. Acad. Sci USA, 93:1988-1992 (1996)). The immunoprecipitates werethen subjected to western gel/blot analysis using the 4G 10anti-phosphotyrosine monoclonal antibody (UBI, Lake Placid, N.Y.).Immunoreactive bands were visualized with an ABC kit according tomanufacturers directions (Vector Laboratories).

To establish stable cell lines, each of the chimeric constructs wasco-transfected with a plasmid containing the neomycin resistance genevia calcium phosphate precipitation into NIH 3T3 cells. Clonesproliferating in the presence of G418 were screened for their ability tobind to VEGF. Clones expressing either the flt-1(1,2,3)/FLT4 orflt-1(2)/FLT4 chimera were analyzed in a cell binding assay to determinethe Kd for VEGF by titrating a trace amount of ¹²⁵I-VEGF₁₆₅ (approx.5000 cpm/ml final) with increasing amounts of cold VEGF₁₆₅. First theadherent cells were washed with cold binding buffer C (DMEM/F12 mediawith 0.2% BSA and 25 mM HEPES, pH 7.4), then ¹²⁵I-VEGF₁₆₅ and the coldcompetitor, each in 0.5 mls buffer C, were added simultaneously. Thecells were then placed at 4° C. for 4 hours. After aspirating off thebinding buffer, the cells were washed with cold PBS and then twice withcold PBS containing 2M NaCl. Finally, the cells were lysed with 0.25MNaOH and the entire lysate was counted in a gamma counter. Results wereanalyzed and the Kds calculated using the Scatchard analysis program NewLigand (Genentech, Inc.).

NIH 3T3 cells stably expressing either the flt-1(1,2,3)/FLT4 or theflt-1(2)/FLT4 chimeric receptors were plated in 12-well format at 50,000cells/well in low glucose DMEM media containing 10% FBS, 100 units/mlPenicillin-Streptomycin (Gibco BRL), 2 mM Glutamine, 2.5 microgram/mlFungizone (Gibco BRL), and 200 micrograms/ml G418 (Gibco BRL). Following18-24 hours of serum starvation in media containing 0.5% FBS, growthfactors or 10% FBS were added. The concentration of VEGF₁₆₅ added rangedfrom 5 pg/ml to 300 ng/ml; PIGF₁₅₂ concentrations were between 5.12ng/ml and 3.2 micrograms/ml; the concentration of VEGF-C was 40 ng/mland 4 micrograms/ml. Following stimulation for 12-16 hours at 37° C.,[³H]thymidine (1 mCi/ml; 5 Ci/mmol) was added for a final concentrationof 1 microcurie/ml and incubation proceeded at 37° C. for 4 hours.Removal of the media and several PBS washes was succeeded by TCAprecipitation. Following the removal of TCA, cells were then lysed with0.2N NaOH, 1% SDS, transferred to scintillation vials and neutralizedwith 2M Na₂OAc, pH 4.0. The samples were counted using the tritiumchannel.

The results of these experiments demonstrated that while VEGF did notstimulate tyrosine phosphorylation in cells transiently expressing theintact FLT4 receptor, significant tyrosine phosphorylation was observedin cells transiently expressing the fit-1(2)/FLT4 chimeric receptor orthe flt-1(1,2,3)/FLT4 chimeric receptor. Thus, these experimentsdemonstrate that the flt-1(2)/FLT4 chimeric receptor and the fit-1(1,2,3)/FLT4 chimeric receptor are able to bind and specifically respond toVEGF. Moreover, these clones showed a significant response to VEGF inthe thymidine incorporation assay.

Concluding Remarks:

The foregoing description details specific methods which can be employedto practice the present invention. Having detailed such specificmethods, those skilled in the art will well enough known how to devisealternative reliable methods at arriving at the same information inusing the fruits of the present invention. Thus, however, detailed theforegoing may appear in text, it should not be construed as limiting theoverall scope thereof; rather, the ambit of the present invention is tobe determined only by the lawful construction of the appended claims.All documents cited herein are expressly incorporated by reference.

1-28. (canceled)
 29. A protein comprising a heterologous polypeptidefused to a first Ig-like domain and a second Ig-like domain, whereinsaid first Ig-like domain comprisesPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQT (SEQ ID NO: 49) orPFIASVSDQHGVVYITENKNKTVVIPCLGSISNLNVSLCARYPEKRFVPDGNRISWDSKKGFTIPSYMISYAGMVFCEAKINDESYQSIMYIVVVVG (SEQ ID NO: 50) and said secondIg-like domain comprisesVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVHIYDK (SEQ ID NO: 51) orVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK (SEQ ID NO: 52), whereinsaid first Ig-like domain and said second Ig-like domain are present inorder in said protein and wherein said protein (a) comprises fewer than7 Ig-like domains; and (b) binds to VEGF.
 30. The protein of claim 29further comprising a first peptide between said first Ig-like domain andsaid second Ig-like domain and wherein said first peptide comprisesNTIID (amino acids 227 to 231 of SEQ ID NO: 1) or YRIYD (amino acids 221to 225 of SEQ ID NO: 2).
 31. The protein of claim 30 further comprisinga third Ig-like domain, wherein said third Ig-like domain is present insaid protein before said first Ig-like domain and wherein said thirdIg-like domain comprisesPELSLKGTQHIMQAGQTLHLQCRGEAAHKWSLPEMVSKESERLSITKSACGRNGKQFCSTLTLNTAQANHTGFYSCKYLAVPTSKKKETESAIYIFI (amino acids 32 to 128 of SEQ IDNO: 1) or PRLSIQKDILTIKANTTLQITCRGQRDLDWLWPNNQSGSEQRVEVTECSDGLFCKTLTIPKVIGNDTGAYKCFYRETDLASVIYVYV (amino acids 32 to 118 of SEQ ID NO: 2). 32.The protein of claim 31 further comprising a second peptide between saidthird Ig-like domain and said first Ig-like domain and wherein saidthird peptide comprises SDTGR (amino acids 129 to 133 of SEQ ID NO: 1)or QDYRS (amino acids 119 to 123 of SEQ ID NO: 2).
 33. The protein ofany one of claims 29-32, wherein the heterologous polypeptide comprisesan immunoglobulin sequence.
 34. The protein of claim 33, wherein theimmunoglobulin sequence is an immunoglobulin constant domain sequence.35. The protein of claim 33, wherein the immunoglobulin sequence is theFc region of an immunoglobulin heavy chain.
 36. The protein of claim 33,wherein the immunoglobulin sequence comprises functionally active hinge,CH2 and CH3 domains of the constant region of an immunoglobulin heavychain.
 37. The protein of claim 33, wherein the immunoglobulin is IgG1or IgG3.
 38. The protein of claim 34, wherein the immunoglobulin is IgG1or IgG3.
 39. The protein of claim 35, wherein the immunoglobulin is IgG1or IgG3.
 40. The protein of claim 36, wherein the immunoglobulin is IgG1or IgG3.